Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-30
2002-03-19
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06358681
ABSTRACT:
BACKGROUND
Alzheimer's disease (AD) is a debilitating illness that affects millions of Americans. New strategies are beginning to emerge for diagnosis of this condition, a condition that currently can only be diagnosed with certainty at autopsy. If diagnostic strategies can improve, treatment of the disease at an earlier stage, before symptoms emerge, may be possible. In fact, the only drug currently available to treat the symptoms of AD tends to be most effective if given in early stages of disease. Clearly, early and definitive diagnosis is essential.
AD is characterized clinically by cognitive decline and memory loss and neuropathologically by the presence of neurofibrillary tangles (NFTs), neuropil threads (NTs), senile plaques (SPs), and regionally specific neuronal loss (Selkoe, D. J. 1994
Annu. Rev. Cell Biol.
10:373-403; Trojanowski, J. Q. et al. 1996 In:
Current Neurology,
Vol. XVI, Boston: Houghton Mifflin). The gradual accumulation of paired helical filaments composed of abnormal tau in NFTs and NTs (Lee, VM-Y et al. 1991
Science
251:675-678) as well as beta-amyloid-containing fibrils within SPs (Selkoe, D. J. 1994 Annu. Rev. Cell Biol. 10:373-403) have been implicated in the pathogenesis of AD. Similar neuropathological findings also are observed in the brains of elderly Down's syndrome (DS) patients who survive beyond the fourth decade of life.
Although the molecular mechanisms responsible for the pathogenesis of NFTs, NTs, and SPs remain to be clarified, immunohistochemical and Western blot analyses of AD brains have allowed detailed characterization of the abnormal tau, beta-amyloid-containing proteins in these lesions (Arai, H. et al. 1990
Proc. Natl. Acad. Sci. USA
87:2249-2253; Arai, H. et al. 1991.
Ann. Neurol.
30:686-693; Selkoe, D. J. et al. 1986.
J. Neurochem.
46:1820-1834). Congo Red and analogs thereof have been used to characterize amyloid plaques in Alzheimer's brains since this fluorescent dye and analogs thereof bind to peptides that make up the fibrils of these plaques (Ashburn et al. 1996
Chemistry and Biology
3:351-358). Northern blot analyses of AD brains also have identified changes in a variety of mRNAs, including those encoding the amyloid precursor proteins (Kang, J. et al. 1987
Nature
325:733-736; Goldgaber, D. et al. 1987.
Science
235:877-880; Robakis, N. K. et al. 1987.
Proc. Natl. Acad. Sci. USA
84:4190-4194; Tanzi, R. E. et al. 1987.
Science
235:880-884; Golde, T. E. et al. 1990.
Neuron
4:253-267). Furthermore, in situ hybridization histochemistry has localized abnormal tau and amyloid precursor mRNAs to neurons and glia in the normal and AD brain (Tanzi, R. E. et al. 1987.
Science
235:880-884; Golde, T. E. et al. 1990.
Neuron
4:253-267; Kosik, K. S. et al. 1989.
Ann. Neurol.
26:353-361; Bahmanyar, S. et al. 1987.
Science
237:77-88; Schmechel, D. E. et al. 1988.
Alzheim. Dis. Assoc. Disord.
2:96-111). Although other protein components in NFTs and SPs have been identified (Schmidt, M. L. et al. 1994.
Exp. Neurol.
130:311-322; Strittmatter, W. J. and A. D. Roses. 1995.
Proc. Natl. Acad. Sci. USA
92:4725-4727), no data are available which provide information on whether RNAs exist in NFTs and SPs themselves. In fact, little is known about the non-proteinaceous components of SPs and NFTs.
The present invention provides a method for detecting the presence of and identifying RNAs, specifically mRNAs, in NFTs, NTs, and SPs of AD brain tissue. Using this method, it has now been found that neuronal mRNAs predominate in SPs in Alzheimer's disease.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of identifying senile plaques, neurofibrillary tangles and neuropil threads in brain tissue which comprises contacting brain tissue with a fluorescent dye capable of intercalating selectively into nucleic acids and detecting any fluorescence in the brain tissue indicative of senile plaques, neurofibrillary tangles and neuropil threads in the brain tissue.
Another object of the present invention is to provide a method of identifying RNAs in senile plaques, neurofibrillary tangles, and neuropil threads of brain tissue which encode proteins involved in the pathogenesis of Alzheimer's disease which comprises isolating single senile plaques in brain tissue by immunocytochemical techniques; identifying the presence of RNA by contacting said senile plaque with a fluorescent dye capable of intercalating selectively into nucleic acids; amplifying the identified RNA; and determining whether the amplified RNA product hybridizes to any known cDNAs for proteins involved in the pathogenesis of Alzheimer's disease.
Yet another object of the present invention is a method of diagnosing Alzheimer's disease comprising identifying the presence of RNA encoding a protein known to be involved in the pathogenesis of Alzheimer's disease.
DETAILED DESCRIPTION OF THE INVENTION
Acridine orange (AO) is a fluorescent dye that intercalates selectively into nucleic acids (Schummelfeder, N. 1958.
J. Histochem. Cytochem.
6:392-393; von Bertalanffy, L. and I. Bickis. 1956.
J. Histochem. Cytochem.
1956. 4:481-493; Rigler, R. 1966.
Acta Physiol. Scand.
67 (Suppl.):7-122). AO histofluorescence has been used to detect RNA and DNA in malignant tumor cells as well as in cell and tissue homogenates separated by gel electrophoresis (Dart, L. H. and T. R. Turner. 1959.
Lab. Invest.
8:1513-1522; McMaster, G. K. and G. G. Carmichael. 1977.
Proc. Natl. Acad. Sci. USA
74:4835-4838; Pinto, A. et al. 1990.
Arch. Pathol. Lab. Med.
114 (6):585-588). Cytoplasmic and nuclear RNA species also have been visualized in tissue sections of the developing and adult brain by AO histochemistry (Schmued, L. C. et al. 1982.
J. Histochem. Cytochem.
30:123-128; Topaloglu, H. and H. B. Sarnat. 1989.
Anat. Rec.
224:88-93; Mai, J. K. et al. 1984.
J. Histochem. Cytochem.
32:97-104). Although AO also binds to mucopolysaccharides, they are not visualized in brain by AO histochemistry due to their low abundance (Szabo, M. M. and E. Roboz-Einstein. 1962.
Arch. Biochem. Biophys.
98:406-412).
The affinity of AO for nucleic acids is dependent upon the concentration of the dye in the staining buffer and the pH of the solution. A low dye concentration (approximately 10-100 &mgr;g/ml) and a pH of 4.0 allows for the optimal intercalation of AO into RNA and DNA, thereby allowing the in situ visualization of these macromolecules (von Bertalanffy, L. and I. Bickis. 1956.
J. Histochem. Cytochem.
1956. 4:481-493; Dart, L. H. and T. R. Turner. 1959.
Lab. Invest.
8:1513-1522; Mikel, U. V. and R. L. Becker. 1991.
Analyt. Quant. Cytol. Histol.
13:253-260). Specifically, upon excitation with ultraviolet/blue spectra (approximately 470-490 nm wavelength), AO intercalated into RNA emits a bright orange-red fluorescence, whereas AO intercalated into DNA emits a yellowish-green fluorescence (von Bertalanffy, L. and I. Bickis. 1956.
J. Histochem. Cytochem.
1956. 4:481-493; Rigler, R. 1966.
Acta Physiol. Scand.
67 (Suppl.): 7-122). In tissue sections, AO-labeled RNA and DNA stand out against the pale green background of the surrounding neuropil and white matter tracts that lack abundant nucleic acids. AO histochemistry can be used on paraffin-embedded brain sections (Topaloglu, H. and H. B. Sarnat. 1989.
Anat. Rec.
224:88-93; Mai, J. K. et al. 1984.
J. Histochem. Cytochem.
32:97-104), and can be combined with other histochemical/immunohistochemical techniques.
Using AO histochemistry, it has now been determined that cytoplasmic RNA species, including either ribosomal, transfer, or messenger (mRNA), are present in NFTs, NTs, and SPs of brains from AD and DS patients. AO histofluorescence was used to screen the brains of patients with AD and DS, as well as normal controls and non-AD patients with other neurodegenerative disorders, to determine whether cytoplasmic RNA species are detectable within NFTs, NTs, and SPs. In these experiments, the hippocampal formation and the entorhinal cortex were selected for ana
Eberwine James
Ginsberg Stephen
Lee Virginia M.-Y.
Trojanowski John Q.
Jones W. Gary
Licata & Tyrrell P.C.
Souaya Jehanne
The Trustees of the University of Pennsylvania
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